Increased fetal hemoglobin (HbF) levels lessen the severity of symptoms associated with sickle cell disease and beta-thalassemia and increase the quality of life and life span of patients. A complete understanding of the mechanism(s) responsible for developmental regulation of globin gene expression (globin gene switching) is important to develop new therapies to increase HbF. Over 30 years of experimental evidence from multiple laboratories strongly supports the hypothesis that DNA methylation plays a fundamental role in silencing expression of the gamma-globin gene in the adults. The use of pharmacological inhibitors of DNA methyltransferase to increase HbF levels was pioneered in the baboon model in our laboratory and led to a series of clinical trials that demonstrated the effectiveness of these drugs in patients. Using the baboon model, our laboratory also made the novel and important observation showing that the gamma-globin gene promoter was demethylated in a progressive manner during fetal liver erythropoiesis. The mechanism responsible for DNA demethylation of the gamma-globin promoter during fetal liver erythropoiesis remains unknown. Our preliminary data shows that increased levels of 5-hydroxymethylcytosine (5-hmC), a novel modification of 5-methylcytosine (5-mC) recently found in eukaryotic cells that is catalyzed by the TET dioxygenase family, are associated with the gamma-globin promoter in erythroid cells expressing elevated levels of gamma-globin. 5-hmC has been proposed to be a key intermediate in both passive and active mechanisms of DNA demethylation and therefore our preliminary data strongly suggests that 5-hmC is involved in the mechanism responsible for demethylation of the gamma-globin gene during fetal liver erythropoiesis. This proposal will investigate the hypothesis that DNA demethylation of the gamma-globin gene during fetal erythroid differentiation is accomplished through a TET- mediated pathway involving 5-hmC. Validation of this hypothesis will define a crucial mechanism (s) in normal developmental fetal stage-specific activation of gamma-globin expression. We envision that detailed knowledge of the normal, physiological mechanism responsible for gamma-globin gene demethylation during fetal liver erythropoiesis will foster the development of new strategies targeting this mechanism to achieve DNA demethylation and high level activation of the gamma-globin gene to increase HbF in patients.